The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/T-resistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130 Enterobacterales and 32 Pseudomonas aeruginosa isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CT-susceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay.

从政府提交给非媒体的报告中可以明显看出，对快速抗菌药物敏感性的迫切需求。因此，我们开发了一种流式细胞术测定法（FCM），用于直接评估血液中的头孢洛氮烷-他唑巴坦（C / T）敏感性（BC），从标志阳性开始需要不到2小时才能报告。使用接种在BC需氧瓶中的C / T易感和C / T耐药的革兰氏阴性菌（美国Becton-Dickinson）对方案进行了优化，然后针对不同的C / T浓度（1 / 4、2 / 4）进行了优化，4/4和8/4 mg / L）孵育1小时（37°C），然后进行FCM和软件分析。使用CytoFLEX细胞计数器（Beckman-Coulter，美国），荧光膜通透性和膜电位染料被比较地用于检测早期细胞损伤。重复性，再现性，测定BC阳性后48小时的测定的稳定性和稳定性。内部验证是在加标有130瓶的BC瓶中进行的肠杆菌和32铜绿假单胞菌来自波尔图大学（葡萄牙）的分离株，包括13种ATCC分离株。此外，还测试了在西班牙马德里的Ramon y Cajal医院从BC阳性的细菌中检出的64克阴性杆菌。比较FCM和肉汤微量稀释结果，计算出分类一致性（CA）和分析误差。只有膜电位染料才能清楚地区分CT敏感和CT抵抗分离株。观察到极好的可重复性，可重复性和方法间的一致性。总体而言，使用带有2个主要错误的EUCAST标准，CA为99.1％，使用带有2个主要和1个较小错误的CLSI标准，CA为98.7％。一种用于测试C / T敏感性的新的，准确的，超快速的FCM（<2小时）可提供准确的结果，并将扩展当前的FCM抗菌药物敏感性测定。